The release of neurotransmitters at synapses is central to information processing in the nervous system. This process is altered in many psychiatric and neurological disorders, and the genes encoding the protein machinery have been repeatedly implicated in anxiety, depression, and schizophrenia in humans and in rodent models. Even though the responsible protein machinery has been known for some time, it is still not understood how the entry of calcium at nerve endings triggers release of neurotransmitters rapidly and synchronously to enable timely correlations of information in nervous systems. In the current project period [NOTE- this grant was at that time entitled Regulation of Exocytosis Studied with Flipped SNAREs and the title was changed in this application to more precisely reflect the current technical approaches.] we have obtained an X-ray crystal structure that appears to represent a pre-fusion intermediate in which the synaptic vesicle is docked and clamped by Complexin half-way thru the zippering process of the SNARE complex, based on correlative biochemical and genetic studies and functional reconstitution. These studies have suggested an innovative structural/biochemical working model that for the first time can explain in a concrete manner how synchronous release of transmitters occurs. In this competing renewal, we now propose to probe this model and its implications deeply, and as a result we expect to gain fundamental insights into synaptic transmission, whether the starting model is fully accurate or whether it needs to be modified as a result. The focus will be the exciting, and entirely unexpected, co-operative zig-zag array of half-zippered SNARE complexes revealed by the crystal structure in which a Complexin bound to one SNARE complex in a novel 'open' conformation inhibits zippering of a second SNARE complex in trans via its 'accessory' helix. We aim to establish the functional, compositional, and dynamic properties of the array as it occurs on lipid bilayers and at vesicle-bilayer junctions, and how the calcium sensor Synaptotagmin releases this clamped complex to trigger exocytosis, using mainly single event/single molecule optical techniques in completely defined systems and with genetic correlations in Drosophila to valid the physiological significance of key findings when possible. PUBLIC HEALTH RELEVANCE: The release of neurotransmitters at synapses is central to information processing and is altered in many psychiatric and neurological disorders. Even though the responsible protein machinery has been known for some time, it is still not understood how the entry of calcium at nerve endings triggers release of neurotransmitters rapidly and synchronously. We have developed and propose to test an innovative structural/biochemical working model that for the first time can explain in a concrete manner how synchronous release of transmitters occurs.